The poxviruses are DNA-containing viruses that replicate in the cytoplasm of eukaryotic cells and are pathogenic to many animal species. The poxviruses known to cause human disease include variola, the causative agent of smallpox, molluscum contagiosum, an opportunistic pathogen often infecting AIDS patients, and monkeypox. Gene expression in vaccinia virus, the prototypic member of the poxvirus family, is temporally regulated and can be divided into early, intermediate, and late phases. All three phases of gene expression rely on multiple virally-encoded factors and a multisubunit RNA polymerase with homology to eukaryotic RNA polymerase II. One factor needed for late transcription in vitro has been partially purified from infected cells and designated VLTF-X. Recently, transcription complementation assays were used to demonstrate that VLTF-X activity is present in the cytoplasm and nucleus of uninfected HeLa cells. VLTF-X activity from uninfected cells is indistinguishable from that recovered from infected cells by a variety of biochemical criteria, leading to the hypothesis that VLTF-X is a factor provided by the host cell. Also, a late promoter DNA-binding activity co-purifies with VLTF-X, suggesting that the biochemical role of this factor may be in late promoter recognition. The experiments of this proposal were designed to identify this factor, define its biochemical role in vaccinia virus late transcription, and to define how the protein functions through a comprehensive mutagenesis analysis. These objective will be accomplished by: (1) cloning the gene encoding VLTF-X either by extensive purification of the factor followed by identification of the purified proteins or by using late promoter-containing oligonucleotides to screen an expression library (2) mapping contacts between VLTF-X and DNA through a variety of chemical and enzymatic techniques (3) defining all of the proteins participating in the late transcription system and defining protein-protein contacts and (4) studying the function of VLTF-X through mutagenesis of the protein. These studies have the potential to uncover a previously unidentified role for the host cell in poxvirus infections. Also, it is expected that knowledge of the transcriptional processes of the virus will increase its application as a vector for gene expression and vaccine use.